The ubiquitin/26S proteasome pathway is a fundamental strategy for eukaryotic cell regulation in which proteins are targeted for destruction by assembly on them of specific polyubiquitin degradation signals that are recognized by the 19S regulatory complex of the 26S proteasome. A super family of ubiquitin-like proteins that undergo parallel but distinct conjugation reactions have co-evolved to serve regulatory functions distinct from targeted degradation. The functional specificities of these post-translational modifications require precisely directed protein interactions among components of the ligation machinery. During the current funding period conventional biochemical and genetic approaches have been exploited in part to define the protein interactions at high resolution. Studies proposed for the next funding period comprise of three Specific Aims designed to build upon these advances. Specific Aim 1 will use biochemical and genetic approaches to study E1-catalyzed activation of Class 1 ubiquitin-like proteins and will examine the role of the carboxyl terminal 2-grasp domain as a specificity filter for cognate E2/Ubc recognition, the role of 2-grasp domain versus Ubc12 amino-terminal peptide binding in transition state stabilization during Nedd8 activation, and characterize selected E1 residues in binding versus catalytic roles. Specific Aim 2 will examine the mechanism of a novel subset of Bi-functional Ligases capable of modifying their cognate protein substrates with either ubiquitin to target degradation or the interferon- induced ISG15ubiquitin-like protein to block degradation. Kinetic studies will quantify the ability of UbcH7 (ubiquitin specific) and UbcH8 (ISG15 specific) to support Epf/Trim25 ligase in regulating levels of the anti-mitotic 14-3-3C protein implicated in breast and prostate cancers. Other studies will examine the consequences of ubiquitin versus ISG15 modification on the half life of 14-3-3C. Specific Aim 3 will continue studies on the enzymology of Mdm2 and MdmX homo- and heterodimers as ubiquitin ligases. Kinetic studies will examine the mechanism of MdmX-stimulated Mdm2-dependent ubiquitination, test a two-site Proximal Indexation model for polyubiquitin chain formation by Mdm2, and examine the in vitro conjugating activity of MdmX with Ku70 and Ku80, bona fide substrates of this ligase. These studies are designed to extend our understanding of the mechanism of ubiquitin-dependent targeting for 26S proteasome-mediated degradation and to characterize a new regulatory strategy involving a small subset of Bi-functional Ligases that can provide a functional context for the evolution of the ISG15 ligation pathway in higher eukaryotes. The latter goal is anticipated to define the mechanism for a previously unknown role for interferon-dependent regulation.Modification of cellular proteins with ubiquitin and related ubiquitin-like proteins constitutes a fundamental regulatory strategy for organisms, derangement of which is implicated in a large cohort of inherited and acquired diseases. This funding supports work to understand the underlying principles of this regulatory mechanism and seeks to explicate one pathway by which interferon exerts its anti-viral and immunomodulatory effects that can be exploited therapeutically without the debilitating side effects of the cytokine.